Intracellular pathogens have co-evolved with their hosts to develop multiple mechanisms and strategies to hijack, subvert and utilise the many cellular processes of their unwilling host to facilitate their entry, replication, survival and cell-to-cell spread. Understanding how pathogens take advantage of their host offers the promise of obtaining fundamental insights into basic cellular processes that are frequently deregulated during pathogenic situations. It also provides important insights into the underlying cause of disease and helps identify potential targets for therapeutic intervention. Our research uses a combination of quantitative imaging and biochemical approaches to study Vaccinia virus as a model system to interrogate the regulation and function of Src and Rho GTPase signalling, cytoskeletal dynamics, actin and microtubule-based transport as well as cell migration. Outside the context of Vaccinia infection, we investigate the cellular function of the focal adhesion protein Tes, a tumour suppressor that negatively regulates Mena-dependent cell migration. In addition, we also examine how different subunit isoforms impact on the actin nucleating activities and cellular function of Arp2/3 complex family members, as well as, the mechanisms regulating the assembly and function of invadopodia.
The actin cytoskeleton provides the driving force and structural support for the physical integrity of cells, and a wide range of fundamental cellular processes such as membrane trafficking and cell migration. Unfortunately, the actin cytoskeleton also helps promote the entry and spread of many different intracellular pathogens.
During Vaccinia virus infection, newly assembled virions fuse with the plasma membrane to exit the cell. Not all virions, however, are immediately released, as some remain attached to the outside of the cell. These cell-associated enveloped virus (CEV) induce Arp2/3-dependent actin polymerization to enhance their spread into adjacent cells. CEV achieve this by stimulating Src and Abl kinase driven phosphorylation of A36, an integral viral membrane protein localized beneath CEV. Phosphorylation of A36 leads to the recruitment of a signalling network consisting of Cdc42, Intersectin, Grb2, Nck, WIP and N-WASP that locally activates Arp2/3-mediated actin polymerization.
Vaccinia stimulates actin polymerisation in a highly localized, stereotypic and robust fashion. This makes the virus a powerful model to understand how this important signalling network stimulates Arp2/3-mediated actin polymerisation. This includes detailed knowledge of how protein interactions and dynamics impacts on the output of the system as well as the properties of the different Arp2/3 complex family members. Moreover, the information and reagents we generate are relevant for other Arp2/3 driven processes such as invadopodia formation and cell migration.
Microtubule-based transport is the primary way in which cellular cargoes are moved micron distances in a directed fashion. It is perhaps not surprising then, that viruses have developed numerous strategies to use this rapid and efficient cellular transport system. Moreover, the ability of large viruses, such as Vaccinia, to hijack microtubule transport is essential, as their size precludes their movement by diffusion. Examining how viruses use microtubules and their associated motors will help us to understand how infection is established, as well as, the mechanisms underlying viral assembly and spread. It also promises to uncover fundamental insights into the molecular basis of microtubule motor recruitment and regulation.
During Vaccinia virus egress, intracellular enveloped virus (IEV) particles are transported on microtubules from their perinuclear site of assembly to the cell periphery by kinesin-1. The interaction of the kinesin light chain (KLC) TPR repeats with a bi-partite tryptophan motif in A36, an integral viral membrane protein recruits the motor to IEV. Related bi-partite tryptophan motifs capable of binding the KLC TPR repeats are also found in over 300 human proteins.
We are currently reconstituting microtubule-based transport of Vaccinia in vitro as well as using advanced live cell imaging approaches to obtain a complete molecular understanding of how Vaccinia recruits kinesin-1 including determining the relationship between motor number and dynamics and the rate and extent of virus transport.
RhoA-mediated regulation of myosin-II in the actin cortex controls cell contraction and blebbing during 3D cell migration and tumour cell invasion. Cell contraction and blebbing also frequently occur as part of the cytopathic effect seen during many viral infections. We recently demonstrated that the Vaccinia virus protein F11, which localises to the plasma membrane, is required for virus induced cell contraction early during infection.
F11 also induces migration of infected cells by inhibiting RhoA signalling by interacting directly with RhoA. This inhibition enhances viral release and promotes the spread of infection by increasing microtubule dynamics and modulating the actin cortex. F11 inhibits RhoA by functioning as a PDZ containing scaffolding protein that binds the PDZ binding motif of the RhoGAP, Myosin-9A. F11 is the first example of a viral protein containing a functional PDZ domain.
Curiously, F11-induced cell contraction depends on RhoC and not RhoA signalling to ROCK. Moreover, RhoC driven contraction requires the upstream inhibition of RhoD signalling by F11. This inhibition prevents RhoD from regulating its downstream effector Pak6, alleviating the suppression of RhoC by the kinase. Our observations demonstrate that RhoD recruits Pak6 to the plasma membrane to antagonise RhoC signalling. Ongoing work aims to understand how Pak6 antagonises RhoC and examine the function of this new signalling network during tumour invasion, given RhoC is more important than RhoA in promoting cancer metastasis.
DNA DAMAGE RESPONSE
In contrast to the majority of large DNA viruses, poxviruses replicate their genomes in the cytoplasm of infected cells. The mechanism of Vaccinia genome replication is still not understood, but was always thought to be largely independent of host DNA replication machinery. We recently demonstrated that Vaccinia induces cytoplasmic activation of ATR prior to genome uncoating. This is unexpected, as ATR plays a fundamental role maintaining genome integrity in the nucleus during the DNA damage response.
ATR, RPA, INTS7 and Chk1 are recruited to cytoplasmic viral DNA factories and inhibition of ATR signalling suppresses genome replication. RPA and PCNA, which interact with the viral polymerase are also required for genome replication. The ATR activator TOPBP1 also promotes genome replication and associates with the viral replisome. Our data reveal that, in contrast to long held beliefs, Vaccinia uses host DNA replication and repair machinery to amplify its genome.
Our observations profoundly change the way we think about how Vaccinia replicates in the cytoplasm. ATR facilitates eukaryote genome replication in response to ssDNA breaks and replication stress in a process that is still not fully understood. Vaccinia now provides a new model to obtain additional insights into how ATR functions. We are currently investigating the nature of the Vaccinia replisome and how ATR is activated independently of incoming viral DNA to promote genome replication.
SEPTINS AND CLATHRIN
During its egress Vaccinia transiently recruits AP-2 and clathrin after it fuses with the plasma membrane. This recruitment enhances virus induced actin polymerisation by promoting clustering of A36 and N-WASP. AP-2 and clathrin are recruited to the virus by Eps15 and intersectin-1, which interact directly with three NPF motifs in the C-terminus of A36. Loss of these NPF motifs reduces virus release and cell-to-cell spread as the lack of recruitment of the RhoGEF intersectin-1 leads to impaired Cdc42 activation and N-WASP driven Arp2/3 actin polymerization.
We recently found that prior to clathrin, Vaccinia recruits septins after fusion with the plasma membrane. Septins are evolutionary conserved components of the cytoskeleton that participate in a variety of fundamental cellular processes including division, migration and membrane trafficking. Septin depletion increases virus release and spread, as well as virus induced actin polymerization. Septins are displaced from the virus when actin polymerization is initiated. Interestingly, septin loss depends on the recruitment of Nck, but not Arp2/3 driven actin polymerization. Moreover, it is the recruitment of dynamin by Nck together with formin-mediated actin polymerization that leads to septin loss. Our analysis represents the first example where septins play an important, albeit a negative role during virus spread. It also suggests that septin dynamics is regulated by dynamin and formin-mediated actin polymerization.